Selective Perturbation of the Myosin Recovery Stroke by Point Mutations at the Base of the Lever Arm Affects ATP Hydrolysis and Phosphate Release

Andraés Maélnaési-Csizmadia,Judit Toéth,David S Pearson,Csaba Heteényi,Laészloé Nyitray,Michael A Geeves,Clive R Bagshaw,Mihaély Kovaécs

doi:10.1074/jbc.m701447200

Andraés Maélnaési-Csizmadia, Judit Toéth + Show 6 more

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https://doi.org/10.1074/jbc.m701447200

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Abstract

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.

Highlights

Various steps of the myosin mechanochemical cycle are linked to large conformational changes of the motor domain, which contains the actin and ATP binding sites as well as the converter region that forms the base of the extended lever arm domain
Pled to a large rotation of the lever arm, and the conformational rearrangement has been termed the recovery stroke, which constitutes the priming of the myosin head in an actin-detached state
The rate constants of ADP dissociation (k6) were determined by chasing experiments in which the motor domain1⁄7ADP complexes were mixed with high concentrations of ATP in the stopped-flow (Fig. 6B)

Summary

Introduction

Various steps of the myosin mechanochemical cycle are linked to large conformational changes of the motor domain, which contains the actin and ATP binding sites as well as the converter region that forms the base of the extended lever arm domain. The converter/lever arm module is thought to amplify the structural changes occurring at the ATPase active site to produce a large working stroke [1, 2]. Upon interacting with ATP, the motor domain undergoes a crystallographically identified large structural rearrangement before hydrolysis takes place During this transition, the movement of the switch-2 loop of the active site toward the ␥-phosphate of ATP brings catalytically important residues to their active positions. Besides its role in ATP hydrolysis and priming the myosin head, the recovery stroke may be important in fine-tuning the steady-state distribution of myosin molecules in up and down lever arm orientations and, in

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